Nitrite reductase activity in F420-dependent sulphite reductase (Fsr) from Methanocaldococcus jannaschii

Methanocaldococcus jannaschii (Mj), a hyperthermophilic and evolutionarily deeply rooted methanogenic archaeon from a deep-sea hydrothermal vent, produces F420-dependent sulphite reductase (Fsr) in response to exposure to sulphite. This enzyme allows Mj to detoxify sulphite, a potent inhibitor of methyl coenzyme-M reductase (Mcr), by reducing it to sulphide with reduced coenzyme F420 (F420H2) as an electron donor; Mcr is essential for energy production for a methanogen. Fsr allows Mj to utilize sulphite as a sulphur source. Nitrite is another potent inhibitor of Mcr and is toxic to methanogens. It is reduced by most sulphite reductases. In this study, we report that MjFsr reduced nitrite to ammonia with F420H2 with physiologically relevant K m values (nitrite, 8.9 µM; F420H2, 9.7 µM). The enzyme also reduced hydroxylamine with a K m value of 112.4 µM, indicating that it was an intermediate in the reduction of nitrite to ammonia. These results open the possibility that Mj could use nitrite as a nitrogen source if it is provided at a low concentration of the type that occurs in its habitat.


Purification of MjFsrI
The protein was purified anaerobically from Mj cells grown with sulphite as a sulphur source via a previously reported procedure [15,24] but with the following modifications. Cell extracts were fractionated via precipitation with ammonium sulphate and gravity flow-based column chromatography conducted at room temperature (~25 °C) and inside an anaerobic chamber filled with a mixture of N 2 and H 2 (96 : 4, v/v). The (NH 4 ) 2 SO 4 and NaCl solutions were prepared in 25 mM potassium phosphate buffer, pH 7 (buffer A). All chromatography resins were obtained from Cytiva, except F 420 -Sepharose which was prepared in the laboratory [15,25,26]. Recovery of the enzyme was followed by use of an assay for F 420 -dependent sulphite reductase (Fsr) as described below. The first step of purification was treatment of an Mj cell extract with (NH 4 ) 2 SO 4 at 60 % saturation, and activity was found in the supernatant, which was fractionated over a 1×20 cm phenyl-Sepharose column with 6 ml resin. After sample application, the column was washed with five solutions of (NH 4 ) 2 SO 4 at the following concentrations, and these were applied in the sequence as presented: 1, 0.75, 0.5, 0.25 and 0 M. The volume of each wash was 12 ml, except that of the first (1 M) which was 36 ml. The eluates from the 0.25 M (NH 4 ) 2 SO 4 wash contained Fsr activity and these were pooled and loaded onto an F 420 -Sepharose column [15,25,26] (1×10 cm; 4 ml resin). After sample application, the column was washed first with 28 ml of buffer A and then five aliquots of 8 ml NaCl solutions at the following NaCl concentrations: 0.1, 0.2, 0.3, 0.4 and 0.5 M. Most of the activity was found in the 0.3 M NaCl fractions, which were pooled. The resulting preparation was fractionated on a 1×20 cm column packed with 6 ml QAE-Sephadex resin. Then, the column was washed with 36 ml of buffer A and 12 ml aliquots of four solutions with NaCl at the following concentrations: 0.25, 0.5, 0.75 and 1 M. The 0.25 M NaCl fractions contained Fsr activity and were pooled. SDS-PAGE showed that this final preparation contained an apparently homogeneous enzyme preparation.

Enzyme activity and protein assays and SDS-PAGE
The assays for FNiR, Fsr and F 420 H 2 -dependent hydroxylamine reductase were performed anaerobically using reduced F 420 (F 420 H 2 ) as the electron donor and respective electron acceptors, which were nitrite, sulphite and hydroxylamine, following a previously described procedure [15,17,27]. Briefly, this procedure involved spectrophotometric monitoring of the oxidation of F 420 H 2 at 400 nm, and an extinction coefficient value of 25 mM −1 cm −1 for F 420 was used to calculate the reaction rate [28]. The standard assay employed a reaction mixture with the following components in a total volume of 0.8 ml, and all assays were initiated by enzyme addition: 100 mM potassium phosphate buffer, pH 7; 40 µM F 420 H 2 ; 500 µM sodium nitrite or 1 mM sodium sulphite or 500 µM hydroxylamine. In the assays that were used to determine the kinetic constants [15], the concentration of the relevant substrate was varied.

Iron and acid-labile sulphur content determination
The contents of iron and acid-labile sulphur of MjFsrI were determined as described previously [17], except a solution containing 71.8 µg ml −1 protein in 25 mM potassium phosphate buffer, pH 7, was used. The ammonia produced in the Fsr reaction was estimated using a glutamate dehydrogenase-based assay [15,17,29] employing an AA0100 Kit (Sigma-Aldrich).

uV-visible spectroscopy and HPLC analysis of the flavin component of MjFsrI
The UV-visible spectrum of a 300 µl anaerobic solution of MjFsrI containing 21 µg of homogeneous protein in 25 mM potassium phosphate buffer, pH 7, and 250 mM NaCl was obtained at 25 °C using a Beckman Coulter DU800 spectrophotometer as described previously [17].
The type of flavin present in MjFsrI was identified and its amount was determined via non-degradative extraction followed by HPLC analysis at room temperature via an established method [17], but with modifications. A 400 µl solution of 28.7 µg purified protein in 25 mM potassium phosphate buffer, pH 7, was used and the filtered extract was concentrated by evaporation under a flow of nitrogen before analysis.

Impact Statement
Nitrite is highly toxic to methanogens and the deep-sea hydrothermal vent environment provides a constant supply of this oxyanion at low concentrations. This environmental situation calls for a nitrite detoxification tool in vent methanogens. Here, the occurrence of F 420 -dependent nitrite reductase (FNiR) activity in F 420 -dependent sulphite reductase (Fsr) opens the possibility of vent methanogens employing this enzyme to detoxify nitrite as well as to generate ammonia for cell biosynthesis.

RESuLTS
Before embarking on investigating new activity of MjFsrI, we examined the purified MjFsrI for the core properties that were established at the time of its discovery [15]. In SDS-PAGE, the protein exhibited three bands at ~16, ~45 and ~69 kDa (Fig. 1a), where the last value matched the theoretical subunit size of MjFsrI, which is 69.79 kDa. The ~16 and ~45 kDa bands are known to originate from the degradation of this protein during the sample preparation for SDS-PAGE [15]. The purified enzyme displayed a UV-visible spectrum typical of sirohaem in a low-spin ferric state with peaks at 280, 390 and 590 nm (Fig. 1b) [15,30]. Following this validation, we investigated the new properties of the protein.

Structural and spectroscopic characteristics of MjFsrI
Reversed-phase HPLC identified the flavin extracted from MjFsrI as FAD (Fig. 1c, d). It was estimated that MjFsrI carried 0.52 mol of this molecule per mole of subunit, which suggested that a dimer of the protein assembled one FAD molecule. Chemical assays showed that MjFsrI held 23.81±1. 16

nitrite reduction activity in MjFsrI and kinetics of the reaction
MjFsrI catalysed the reduction of nitrite and hydroxylamine to ammonia by utilizing F 420 H 2 as an electron donor and was not able to utilize NADH or NADPH for these conversions. Accordingly, we term this action as FNiR activity. At a fixed concentration of 300 µM for nitrite and a concentration range of 2-80 µM for F 420 H 2 , the apparent K m for F 420 H 2 was 9.72±1.7 µM and the apparent maximum velocity (V m ) value was 20.3±0.96 µmol F 420 H 2 oxidized or 40.6±1.92 µmol electrons transferred per minute per milligram enzyme (Fig. 2a). Similarly, with 40 µM F 420 H 2 and 5-150 µM nitrite, the apparent K m for nitrite was estimated to be 8.9±0.9 µM and the V m was 18.4±0.36 µmol of F 420 H 2 oxidized or 36.8±0.72 µmol electrons transferred per minute per milligram enzyme (Fig. 2b). A kinetic analysis at 40 µM F 420 H 2 and 25-600 µM hydroxylamine showed that the apparent K m for hydroxylamine was 112.4±14.4 µM and the respective V m was 45.3±1.9 µmol of F 420 H 2 oxidized or 90.6±3.8 µmol electrons transferred per minute per milligram enzyme (Fig. 2c). From an average of the values for the amounts of products in three independent 30 min FNiR reactions with MjFsrI, the enzyme produced 0.0427±0.0005 µmol F 420 and 0.0133±0.0051 µmol ammonia in an assay with 0.08 µmol F 420 H 2 and 0.40 µmol nitrite; in effect, 0.043 µmol F 420 H 2 was consumed or 0.086 µmol electrons was made available for nitrite reduction. Since the production of 1 mol nitrite to ammonia would require 6 mol electrons or 3 mol F 420 H 2 , and the enzyme produced F 420 and ammonia at a ratio of 3 : 1, in the FNiR reaction, about 94 % of the consumed reducing equivalents was recovered in the product.

DISCuSSIon
Fsr of Mj exhibited FNiR activity. This observation could be rationalized thermodynamically and it is also consistent with the chemical mechanism established for other sulphite reductases. Since F 420 H 2 with a mid-point redox potential (E 0 ′) value of −360 mV (see equation 1 below) is an effective reductant for Fsr-catalysed sulphite reduction (equations 2 and 4), thermodynamically the enzyme would be able to utilize F 420 H 2 for nitrite reduction (equations 3 and 5). Studies with Archaeoglobus fulgidus (Af) dissimilatory sulphite reductase (Dsr) have shown that the transformation of sulphite to sulphide involves the formation of enzyme-bound SO 2 − and SO − as intermediates which are stabilized by a set of basic residues (Arg 98 , Arg 170 , Lys 211 and Lys 213 ) [21,32]. The SO 2 − intermediate is structurally similar to NO 2 − and AfDsr indeed reduces nitrite using the above-mentioned structural units [21,32]. The Arg 98 , Arg 170 , Lys 211 and Lys 213 of AfDsr are fully conserved in MjFsrI (Arg 355 , Arg 423 , Lys 460 and Lys 462 [15,31]) and therefore MjFsrI employed a common chemical mechanism for reducing sulphite and nitrite; the recently available crystal structure of MjFsrI indeed shows that Arg 355 , Arg 423 , Lys 460 and Lys 462 are involved in the binding of sulphite and two water molecules at the active site of this protein [31]. The ability of MjFsrI to reduce hydroxylamine (NH 2 OH) indicated that the Fsr-catalysed reduction of nitrite proceeded through the intermediate formation of hydroxylamine and this sequence is also seen with the other sulphite/nitrite reductases [17,18,20,21]. The enzyme was efficient in the utilization of the F 420 H 2 -derived reducing equivalents as it exhibited almost 100 % recovery of this resource into ammonia, the product. These data indicated that NH 2 OH, a reaction intermediate, did not accumulate and this finding is consistent with the observation that the rate of hydroxylamine reduction was more than double of that for the overall nitrite reduction reaction. The higher K m value for hydroxylamine did not pose a problem for this conversion as this intermediate was probably not released from the enzyme.
The K m value of MjFsrI for nitrite (~9 µM) was comparable to a value that has been reported for sulphite (12 µM) and a similar case was found for the specific activities of this enzyme (μmol electrons transferred min -1 mg -1 ): 37 for nitrite reduction (this study) and 32 for sulphite reduction [15]. Thus, the nitrite reduction activity of MjFsrI could be physiologically relevant in Mj with in vivo roles in nitrite detoxification and deriving nitrogen nutrition from this oxyanion. We elaborate on these two possibilities below.
As mentioned in the Introduction, MjFsrI acts as a sulphite detoxification enzyme and allows Mj to use this toxic compound as a sulphur source [13,15]. Similar to sulphite, nitrite is highly toxic to methanogens as it oxidizes the Ni(I) centre of coenzyme F 430 , a prosthetic group of Mcr [1], an essential enzyme for energy production in these organisms [3]; at a concentration of 50 µM, nitrite fully inactivates purified Mcr in 15 min [1]. In the natural habitat of Mj, a segment of a deep-sea hydrothermal vent, mixing of extremely hot and anaerobic vent water with oxygen-containing cold seawater brings the temperature to a level that is conducive for living organisms [33]. At this locale, the nitrite concentration is kept at a very low level due to chemical reoxidation to nitrate [34]. However, for a constant albeit low-level supply of this oxyanion and high sensitivity of Mcr towards it, it would be advantageous for a vent methanogen, such as Mj, to carry a nitrite detoxification tool, and with the observed high activity and low K m value for nitrite (~ 9 µM), MjFsrI could satisfy this need.
The highest nitrite concentration in a deep-sea hydrothermal vent is never more than 4 µM [34,35]. Based on the data in Fig. 2b, at this nitrite concentration, MjFsrI will provide an FNiR activity of about 6 µmol F 420 H 2 oxidized per minute per milligram protein, which would translate to an ammonium production rate of 2 µmol min -1 mg -1 protein. The doubling time of Mj is about 26 min [36], which corresponds to a maximum growth rate of 1.6 h −1 or 1.6 g of daughter cells per 1 g of mother cell mass per hour. Also, nitrogen constitutes about ~15 % of the cell mass [37]. Thus, to produce 1.6 g of new cells per hour, 1 g of mother cells needs to generate 0.24 g of usable nitrogen or 0.29 g or 0.017 mol ammonia per hour. As estimated from the published data ( Fig. 2a of [15]), under sulphite induction, Fsr constitutes about 10 % of the total cell proteins; 50 % of the dry weight of a cell is due to proteins [38]. This means that 1 g of mother cells could contain up to 50 mg of Fsr protein, and consequently, could produce 0.006 mol ammonia per hour. Therefore, in a hydrothermal vent environment, the FNiR activity of MjFsrI would allow Mj to utilize available nitrite as a sole nitrogen source to maintain about one-third of its maximal growth rate as determined in a laboratory [36] if the nitrogen is the only limiting nutrient. A similar role has also been proposed recently for FsrII in an ANME from marine methane seep sediment [17]. Thus, the Fsr group broadly protects Mcr of marine methanogens and certain ANMEs from sulphite and nitrite inhibition and provides sulphur and nitrogen nutrition to these organisms by reducing these oxyanions.
In summary, the protein that has been described as MjFsrI [15] was shown to provide an FNiR function with physiologically relevant kinetic properties without causing toxicity of hydroxylamine, a reaction intermediate. A calculation based on the kinetic properties of the enzyme, growth kinetics of the organism, and known concentrations for nitrite in the hydrothermal vent fluid showed that the FNiR activity of MjFsrI would support a reasonably high growth rate for Mj with nitrite as the sole nitrogen source if it is induced under in situ conditions [15]. This assessment made for a rather low concentration of nitrite, a toxic oxyanion for the methanogens, sets the stage for growth studies with Mj and other vent methanogens with nitrite as the sole nitrogen source in a continuous culture system. Here a steady supply of nitrite at a low concentration would provide an ecologically relevant environmental condition.

If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines? Yes
Author response to reviewers to Version 1 We thank the reviewers for thoughtful and detailed reviews of our manuscript and for offering valuable suggestions. These have helped us to improve the manuscript. We present below our responses to each of the comments.

The cited line numbers for the revised manuscript refer to the file without tracks.
Responses to the comments from Reviewer 1 Comment 1.The manuscript by Heryakusuma et al. describes the ability of an F 420 -dependent sulfite reductase (Fsr) from M. jannaschiito reduce nitrite to ammonium, which was supported by a nitrite reductase activity assay. Based on this finding, the authors predict that the M. jannaschiiFsr may be involved in nitrite detoxification given that the environment where the organism thrives has an infinite availability of this oxyanion. It is widely known that sulfite reductases, as well as nitrite reductases, catalyze both sulfite and nitrite, and this is primarily due to the physicochemical similarities of the two substrates. Given this fact, it does not necessarily follow that a sulfite reductase may be involved in nitrite detoxification or that a nitrite reductase is involved in sulfite detoxification. Therefore, the claim that M. jannaschiiFsr may be involved in nitrite detoxification must be sufficiently substantiated. One way to do this is to perform RT-qPCR to determine the level of transcription of the fsrgene in the presence vs absence of nitrite or prove by using bioinformatics that M. jannaschiidoes not possess a homolog of any known nitrite reductases. I recommend accepting this manuscript if one of the two suggested experiments is accomplished.
Response to Comment 1:We thank the reviewer for this comment, and we have performed one of the suggested experiments and it is a bioinformatics analysis of the M.jannaschiigenome. We found thatM. jannaschiidoes not possess a homolog of known nitrite reductases. Two proteins that show homologies to siroheme nitrite reductases are MjFsr or MjFNiR and a dissimilatory sulfite reductase type protein, and the latter does not allow M. jannaschiito reduce sulfite or nitrite(reference 24in our manuscript and our unpublished data). Since our manuscript is focused on the properties of a purified enzyme, we have not included the results of our bioinformatic analysis.We plan to perform a detailed physiological analysis of the nitrite metabolism of M. jannaschiiin the near future. The purified enzyme displayed a UV-visible spectrum typical of siroheme in low-spin ferric state with peaks at 280, 390, 117 and 590 nm (Fig. 1B) (14, 30)'. The enzyme also contains 6 [4Fe-4S] clusters, which each contribute to the absorbance in the near UV/visible region. Is the Fe-S cluster contribution to the absorbance spectrum known? Presumably the absorption due to FAD is difficult to see amongst the siroheme/Fe-S cluster absorbance? Figure 1C Perhaps I misunderstand what this is, but why is the FAD standard elution time shifted from that of the co-injection sample (and sample extract)? L150

Comment 2.The degradation seen in SDS-PAGE is likely from proteolysis given that the authors did not use any protease inhibi-
From an average of the data from the estimation of products in three independent 30 minutes long FNiR reactions with MjFsrI. This sentence does not make sense; it is lacking a verb. L151 From the data presented, the 3:1 ratio for FADH2 consumed to ammonia produced is what would be expected for the six electron reduction of nitrite. How was the ammonia production determined (I did not see this in the methods)? Also, a rough calculation indicated that the enzyme should be capable of turning over all of the substrate added in 30 min, but it doesn't -only about half of the FADH2 is consumed (limiting substrate). Why is this? I note that the authors in the conclusion section suggest that ~100% of the FADH2 is utilised in the reaction, but 0.043/0.08 is ~53%. The discussion section argues that the nitrite reductase activity of MjFsrI could be physiologically relevant, both for detoxification of nitrite and as a source of nitrogen. There is a rough calculation that supports this based on levels of nitrite and enzyme. In the introduction it is mentioned that several Methanocaldococcus species utilize nitrate as nitrogen source. Does M. jannaschii have this capability? The first step in this process is the reduction of nitrate to nitrite, so nitrite is generated endogenously in such organisms. If this occurs here, then there is all the more reason to need a nitrite reductase. On that note, the authors need to provide further information -what other potential nitrite reductases are present in M. jannaschii?

Please rate the manuscript for methodological rigour Good
Please rate the quality of the presentation and structure of the manuscript Satisfactory

Anonymous.
Date report received: 21 October 2022 Recommendation: Minor Amendment Comments: 1. Methodological rigour, reproducibility and availability of underlying data The authors set up the background that Methanocaldococcus jannaschii is a well studied methanogen. They also make the case that in Mj native habitat, there is a low level but consistent supply of oxidized nitrogen compounds, Finally, they present the premise that an abundant protein Mj-FsrI, previously shown to be a sulfitre reductase, can also act as a nitrite reductase. In fact, this multi-substrate function has been demonstrated with many habitually similar organisms. Excellent experimental setup and rationale for the methods used 2. Presentation of results The results were laid out well. There appears to be another band that runs between 100 kDa and 150 kDa; any speculation about what this band is? It could have benefited from Mass Spec on the isolated protein to validate that was the target, if MjFsrI specific antibodies are non-existant; however, the explanation was thorough enough to be convincing . 3. How the style and organization of the paper communicates and represents key findings The paper is organized well. It is compact and takes the reader from point to point, culminating in MjFsrI has nitrite reduction capability and it could be an advantage in its native habitat. There is no filler in this paper. 4. Literature analysis or discussion The discussion is done well and delves deeply into the estimates of how the enzymatic reduction of nitrite by MjFsrI could be essential for growth in the native habitat. The estimates of the nitrite assimilation from environmental concentrations through cell uptake and enzymatic conversion were compelling. Given how well the bulk of the discussion went, the last sentence (Line 242-243) seemed like an after thought. 5. Any other relevant comments Lines 44-50: Sets up the premise that nitrate/nitrite/hydroxylamine is broadly toxic to methanogens, then says there are pathways certain methanogens utilize to detoxify, or even benefit from, these substrates. Could the premise be that nitrate/nitrite/hydoxylamine processing occurs in a subset of methanogenic organisms which benefit from nitrite? Line 51-57: Similar argument to Lines 44-50. Line 120-121: If the determination that MjFsrI-flavin is FAD, why does the HPLC elution peak in Figure